Process for determining the tumoricidal potential of a sample the use of a nucleic acid which is downregulated in human tumor cells

ABSTRACT

A process for determining whether or not a cancer cell-containing test sample originating from or containing human cells has potential for tumor development, tumor progression or metastasis, wherein the test sample and a second sample originating from non-tumor cells obtained from the same individual or a different individual of the same species are analyzed by incubating each respective sample with a nucleic probe of sequence SEQ ID NO:1 and determining the approximate amount of hybridization of each respective sample with said probe, and comparing the approximate amount of hybridization of the test sample to an approximate amount of hybridization of said second sample to identify whether or not the test sample contains a greater amount of the specific nucleic acid or mixture of nucleic acids than does said second sample.

Carcinogenesis, tumor progression and metastasis result from animbalanced transcriptional program, inappropriate post-translationalmodifications and deregulated epigenetic modifications (Schwirzke, M. etal., Anticancer Res 19 (1999) 1801–1814; Pardee, A. B., Advances inCancer Res 65 (1994) 213–227; Ponta, H., Biochim Biophys Acta 1198(1994) 1–10). Changes of the transcriptional program are due tooncogenes and tumor suppressor genes, fusion proteins created bycytogenetic alterations, altered expression of genes due to unscheduledmethylation by DNA methyltransferases and chromatin modifying enzymessuch as histone acetyltransferases and histone deacetylases (Lin, R. J.et al., Trends Genet 15 (1999) 179–184; Stunnenberg, H. G. et al.,Biochem Biophys Acta 1423 (1999) F15–F33).

For identification of tumor-related candidate genes, transcriptionalprofiling of cellular systems such as metastasizing versusnon-metastasizing cell lines and tumor specimen corresponding todifferent stages of progression is the first step for achievement ofthis goal (Schiemann, S. et al., Anticancer Research 17 (1997) 13–20;Schwirzke, M. et al., Anticancer Research 18 (1998) 1409–1422;Schiemann, S. et al., Clin Exp Metastasis 16 (1998) 129–139). Furthersteps involve analysis of prevalence of the identified alteration indifferent tumors, in-vitro modulation of the gene under consideration byoverexpression and downregulation making use of antisense RNA orribozymes in stable transfectants and assessing the consequences inrelevant in-vitro systems. The advent of nude mouse systems, includingsubcutaneous xenograft systems and orthotopic implantation in which thenatural tropism of metastasis of the tumor under investigation ismaintained, has paved the way for assessment of the functional role ofcandidate genes in vivo (Fidler, I. J., Cancer Metastasis Rev 50 (1986)29–49).

Loss of heterozygosity (LOH) at critical chromocal loci is associatedwith a higher risk of cancer development. LOH at such critical loci maybe used as a valuable marker to determine the potential of cancerdevelopment in early stages and to evaluate the efficacy ofchemopreventive and chemotherapeutic agents. The genes located onchromosome 6q are extremely polymorphic and provide for a naturalheterozygous gene system. LOH on chromosome 6q indicates a highprobability of tumor development such as malignant melanoma (Healy, E.et al., Oncogene 16 (1998) 2213–2218; Robertson, G. P. et al., CancerRes 56 (1996) 1635–1641; Ray, M. E. et al., Oncogene 12 (1996)2527–2533; Millikin, D. et al., Cancer Research 51 (1991) 5449–5453),pancreatic cancer (Griffin, C. A. et al., Cancer Research 55 (1995)2394–2399; cervical cancer (Huettner, P. C. et al., Human Pathol 29(1998) 364–370) prostate cancer (Srikantan, V. et al., Int J Cancer 84(1999) 331–335; MacGrogan, D. and Bookstein, R., Seminars in CancerBiology 8 (1997) 11–19; Verma, R. S. et al., Cancer Investigation 17(1999) 441–447) and breast cancer (Bilanges, B. et al., Oncogene 18(1999) 3979–3988; Chappell, S. A. et al., British J Cancer 75 (1997)1324–1329; Devilee, P., et al., Oncogene 6 (1991) 1705–1711; Noviello,C. et al., Clin Cancer Research 2 (1996) 1601–1606; Fujii, H., et al.,Genes, Chromosomes & Cancer 16 (1996) 35–39).

SUMMARY OF INVENTION

The present invention provides a process for detecting the presence orabsence of at least one specific nucleic acid or mixture of nucleicacids, or distinguishing between two different sequences in a sample,wherein the sample is suspected of containing said sequence orsequences, which process comprises the following steps in order:

-   (a) incubating said sample under stringent hybridization conditions    with a nucleic acid probe which is selected from the group    consisting of:    -   (i) a nucleic acid sequence of SEQ ID NO: 1;    -   (ii) a nucleic acid sequence which is complementary to the        nucleic acid sequence of (i);    -   (iii) a nucleic acid sequence which hybridizes under stringent        conditions with the sequence of (i); and    -   (iv) a nucleic acid sequence which hybridizes under stringent        conditions with the sequence of (ii); and-   (b) determining whether said hybridization has occurred.

Moreover, the present invention provides a process for determiningwhether or not a test sample originating from or containing human cells,preferably a test sample originating from or containing epithelialcells, has potential for tumor development, progression or metastasis ofsaid cells, wherein the test sample and a second sample originating fromnon-tumor cells from the same individual or a different individual ofthe same species is analyzed by:

-   (a) incubating the respective sample under stringent hybridization    conditions with a nucleic acid probe which is selected from the    group consisting of:    -   (i) a nucleic add sequence of SEQ ID NO: 1;    -   (ii) a nucleic acid sequence which is complementary to the        nucleic add sequence of (i);    -   (iii) a nucleic acid sequence which hybridizes under stringent        conditions with the sequence of (i); and    -   (iv) a nucleic acid sequence which hybridizes under stringent        conditions with the sequence of (ii); and-   (b) determining the approximate amount of hybridization of each    respective sample with said probe, and-   (c) comparing the approximate amount of hybridization of the test    sample to an approximate amount of hybridization of said second    sample to identify whether or not the test sample contains a greater    amount of the specific nucleic add or mixture of nucleic acids than    does said second sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the use of the THW gene for diagnostics,especially in the field of cancer. In particular, the invention involvesthe identification and the measurement of the amount of expression ofsaid gene THW in mammalian, especially in malignant tumor cells. Theinvention also relates to diagnosis of the metastatic and progressionpotential of tumor cells.

THW nucleic acid has downregulated expression in tumor cells and iscapable of suppressing tumor progression and/or metastasis, especiallyin malignant melanoma and mammary carcinoma cells.

A tumor suppressor gene named THW is located on chromosome 6q and itsloss or inhibition is correlated with a tumor potential. Also a protein,termed THW, is provided which is downregulated in cancer cells ascompared to their non-cancerous counterparts. THW may be involved intumor suppression and especially in suppression of metastasis. The THWgene codes for a polypeptide of SEQ ID NO:2.

The nucleic acid encoding THW protein is downregulated in tumor cellsand can be selected from the group consisting of:

-   (a) SEQ ID NO: 1;-   (b) a nucleic acid sequence which hybridizes under stringent    conditions with a nucleic acid probe of the complementary sequence    of (a),-   (c) a nucleic acid sequence which, because of the degeneracy of the    genetic code, is not a sequence of (a) or (b), but which codes for a    polypeptide having exactly the same amino acid sequence as a    polypeptide encoded by a sequence of (a) or (b); and-   (d) a nucleic acid sequence which is a fragment of any of the    sequences of (a), (b) or (c).

THW polypeptide is encoded by a nucleic acid selected from the groupconsisting of:

(a) SEQ ID NO: 1;

(b) a nucleic acid sequence which hybridizes under stringent conditionswith a nucleic acid probe of the complementary sequence of (a);

(c) a nucleic acid which is a fragment of any of the sequences of (a) or(b).

Preferably, THW polypeptide has the sequence of SEQ ID NO:2.

The isolated THW polypeptide and, thus, its encoding nucleic acid canoccur in natural allelic variations which differ from individual toindividual. Such variations of the amino acids are usually amino acidsubstitutions. However, they may also be deletions, insertions oradditions of amino adds to the total sequence. The THW protein accordingto the invention—depending, both in respect of the extent and type, onthe cell and cell type in which it is expressed—can be in glycosylatedor non-glycosylated form. Polypeptides with tumor suppressor activitycan be identified by transfection of THW-negative tumor cells withexpression vectors for THW, establishment of stable transfectants andevaluation of their tumoricidal capacity after xenografting into nudemice. Such evaluation can be performed, e.g., according to Boraschi, D.,et al., Cell Immunol. 45 (1979) 188–194 and Boraschi, D., et al., J.Immunol. 131 (1983) 1707–1713.

“Polypeptide with THW activity or THW” means also proteins with minoramino acid variations but with substantially the same THW activity.Substantially the same means that the activities are of the samebiological properties and the polypeptides show at least 90% identity inamino acid sequence.

The term “nucleic acid molecule or nucleic acid” denotes apolynucleotide molecule which can be, for example, a DNA, RNA, orderivatized active DNA or RNA. DNA and/or RNA molecules are preferred,however.

The term “hybridize under stringent conditions” means that two nucleicacid fragments are capable of hybridization to one another understandard hybridization conditions described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (1989) Cold Spring HarborLaboratory Press, New York, USA. More specifically, “stringentconditions” as used herein refer to hybridization in 6.0×SSC at about45° C., followed by a wash. This wash can be with 2.0×SSC at 50° C.Preferably, hybridization is performed using the commercially availableExpress Hyb™ Hybridization Solution of Clontech, which is a non-viscioussolution containing no salmon sperm DNA. The stringency of the saltconcentration in the wash step can be selected, for example, from about2.0×SSC at 50° C., for low stringency, to about 0.2×SSC at 50° C., forhigh stringency. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperatures, about 22°C., to high stringency conditions at about 65° C.

The phrase “nucleic acid or polypeptide” as used throughout thisapplication refers to a nucleic acid or polypeptide having a THWactivity which is substantially free of cellular material or culturemedium when produced by recombinant DNA techniques, or substantiallyfree of chemical precursors or other chemicals when synthesizedchemically. Such a nucleic acid is preferably free of sequences whichnaturally flank the nucleic acid (i.e. sequences located at the 5′ andthe 3′ ends of the nucleic acid) in the organism from which the nucleicacid is derived.

THW can be purified after recombinant production by affinitychromatography using known protein purification techniques, includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocussing, isoelectric focussing, selective precipitation,electrophoresis, or the like.

The polypeptides according to the invention can be produced byrecombinant means, or synthetically. Non-glycosylated THW polypeptide isobtained when it is produced recombinantly in prokaryotes. With the aidof the nucleic acid sequences provided by the invention it is possibleto search for the THW gene or its variants in genomes of any desiredcells (e.g. apart from human cells, also in cells of other mammals), toidentify these and to isolate the desired gene coding for the THWprotein. Such processes and suitable hybridization conditions are knownto a person skilled in the art and are described, for example, bySambrook et al., Molecular Cloning: A Laboratory Manual (1989), ColdSpring Harbor Laboratory Press, New York, USA and Hames, B. D., Higgins,S. G., Nucleic Acid Hybridisation—A Practical Approach (1985) IRL Press,Oxford, England. In this case the standard protocols described in thesepublications are usually used for the experiments.

With the aid of such nucleic acids coding for a THW protein, the proteinaccording to the invention can be obtained in a reproducible manner andin large amounts. For expression in prokaryotic or eukaryotic organisms,such as prokaryotic host cells or eukaryotic host cells, the nucleicacid is integrated into suitable expression vectors, according tomethods familiar to a person skilled in the art. Such an expressionvector preferably contains a regulatable/inducible promoter. Theserecombinant vectors are then introduced for the expression into suitablehost cells such as, e.g., E. coli as a prokaryotic host cell orSaccharomyces cerevisiae, Teratocarcinoma cell line PA-1 sc 9117(Büttner, R., et al., Mol. Cell. Biol. 11 (1991) 3573–3583), insectcells, CHO or COS cells as eukaryotic host cells and the transformed ortransduced host cells are cultured under conditions which allowexpression of the heterologous gene. The isolation of the protein can becarried out according to known methods from the host cell or from theculture supernatant of the host cell. Such methods are described forexample by Ausubel I., Frederick M., Current Protocols in Mol. Biol.(1992), John Wiley and Sons, New York Also in vitro reactivation of theprotein may be necessary if it is not found in soluble form in the cellculture.

The invention further comprises recombinant expression vectors which aresuitable for the expression of THW, recombinant host cells transfectedwith such expression vectors, as well as a process for the recombinantproduction of a protein which is encoded by the THW gene.

The invention further comprises a method for detecting a nucleic acidmolecule of gene THW, comprising incubating a sample (e.g., body fluidssuch as blood, cell lysates or DNA made by reverse transcription ofsample RNA) with the isolated nucleic acid molecule according to theinvention and determining hybridization under stringent conditions ofsaid isolated nucleic acid molecule to a target nucleic acid moleculefor determination of presence of a nucleic acid molecule which is theTHW gene and therefore a method for the identification of the metastaticpotential and/or progression of tumor cells.

To determine whether a cancer cell-containing test sample has potentialfor tumor development, progression or metastasis, the approximate amountof hybridization of the isolated nucleic acid with the target nucleicacid or nucleic acids is determined. The approximate amount ofhybridization need not be determined quantitatively, although aquantitative determination is encompassed by the present invention.Typically, the approximate amount of hybridization is determinedqualitatively, for example, by a sight inspection upon detectinghybridization. For example, if a gel is used to resolve labelled nucleicacid which hybridizes to target nucleic acid in the sample, theresulting band can be inspected visually. One can compare theapproximate amount of hybridization in the test sample to theapproximate amount of hybridization in non-tumor cells. Such non-tumorcells are, e.g., epithelial cells or peripheral blood cells.

As is shown in accordance with the present invention, the THW nucdeicacid is present in a lower amount in a tumor sample than in a samplefree from peripheral blood cells of a healthy donor. A test samplehaving no or low potential for tumor progression or for metastasis willhave a higher amount of the THW nucleic acid of the present inventionthan does a cancer cell sample which has a high tumor progressionpotential or a metastatic potential.

On the basis of the nucleic acids provided by the invention it ispossible to provide a tumor test which uses the detection of THW nucleicacids as a measure of early tumor detection.

Methods of hybridization of a probe and a nucleic acid are known to aperson skilled in the art and are described, for example, in WO89/06698, EP-A 0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879, EP-A0 173 251, EP-A 0 173 251.

In a preferred embodiment of the invention the nucleic acid of thesample is amplified before the test (and reverse-transcribed if thesample nucleic acid is RNA), for example by means of the known PCRtechnique. Usually a derivatized (labeled) nucleic acid probe is usedwithin the framework of nucleic acid diagnostics. This probe iscontacted with a denatured DNA or RNA from the sample which is bound toa carrier and in this process the temperature, ionic strength, pH andother buffer conditions are selected—depending on the length andcomposition of the nucleic acid probe and the resulting meltingtemperature of the expected hybrid—such that the labeled DNA or RNA canbind to homologous DNA or RNA (hybridization see also Wahl, G. M., etal., Proc. Natl. Acad. Sci. USA 76 (1979) 3683–3687). Suitable carriersare membranes or carrier materials based on nitrocellulose (e.g.,Schleicher and Schüll, BA 85, Amersham Hybond, C.), strengthened orbound nitrocellulose in powder form or nylon membranes derivatized withvarious functional groups (e.g., nitro groups) (e.g., Schleicher andSchüll, Nytran; NEN, Gene Screen; Amersham Hybond M.; Pall Biodyne).

Preferably the nucleic acid probe is incubated with the nucleic acid ofthe sample and the hybridization is detected optionally by means of afurther binding partner for the nucleic acid of the sample and/or thenucleic acid probe.

Hybridizing DNA or RNA can be detected by incubating the carrier with anantibody or antibody fragment after thorough washing and saturation toprevent unspecific binding. The antibody or the antibody fragment isdirected towards the substance incorporated during hybridization to thenucleic acid probe. The antibody is in turn labeled. However, it is alsopossible to use a directly labeled DNA. After incubation with theantibodies it is washed again in order to only detect specifically boundantibody conjugates. The determination is then carried out according toknown methods by means of the label on the antibody or the antibodyfragment.

The detection of the expression can be carried out for example as:

-   -   in situ hybridization with fixed whole cells, with fixed tissue        smears,    -   colony hybridization (cells) and plaque hybridization (phages        and viruses),    -   Southern hybridization (DNA detection),    -   Northern hybridization (RNA detection),    -   serum analysis (e.g., cell type analysis of cells in the serum        by slot-blot analysis),    -   after amplification (e.g., PCR technique).

The nucleic acids according to the invention are hence valuableprognostic markers in the diagnosis of the development and progressionpotential of tumors.

The invention further comprises a method for producing a protein whoseexpression is correlated with tumor suppression, by expressing anexogenous DNA in prokaryotic or eukaryotic host cells and isolation ofthe desired protein, wherein the protein is coded by the nucleic acidmolecules according to the invention, preferably by the DNA sequenceshown in SEQ ID NO:1.

The protein can be isolated from the cells or the culture supernatantand purified by chromatographic means, preferably by ion exchangechromatography, affinity chromatography and/or reverse phase HPLC.

The invention further comprises an isolated protein according to theinvention which is encoded by a nucleic acid molecule according to theinvention, preferably having the nucleotide sequence set forth in SEQ IDNO:1.

The present invention relates to the cloning and characterization of thegene THW, which is especially characterized as a tumor suppression gene,and as a downregulated gene indicative of tumorigenic potential.

According to the invention there are provided methods for identifyingand isolation of compounds which have utility in the treatment ofcancer, especially in tumor suppression. These methods include methodsfor modulating the expression of the polypeptides according to theinvention, methods for identifying compounds which can mimic theproteins according to the invention, and methods of identifyingcompounds which can activate said polypeptides and the encoding genes.The methods further include methods for activating the transcription ofTHW gene to mRNA, which preferably downregulates the tumoricidalpotential in a tumor cell. These methods can be conducted in vitro or invivo and may make use of and establish cell lines and transgenic animalmodels of the invention.

THW activity may be measured in several ways. For example, theactivation is apparent by a change in cell physiology, such as increasedmobility and invasiveness in vitro, or by a change in thedifferentiation state, or by a change in cell metabolism leading to anincrease of proliferation.

The THW gene is located on chromosome 6q, a region for which LOH hasbeen found in several tumors, such as malignant melanoma (Healy, E. etal., Oncogene 16 (1998) 2213–2218; Robertson, G. P. et al., Cancer Res56 (1996) 1635–1641; Trent, J. M. et al., Science 247 (1990) 568–71;Ray, M. E. et al., Oncogene 12 (1996) 2527–2533; Millikin, D. et al.,Cancer Research 51 (1991) 5449–5453), pancreatic cancer (Griffin, C. A.et al., Cancer Research 55 (1995) 2394–2399), cervical cancer (Huettner,P. C., et al., Human Pathol 29 (1998) 364–370), prostate (Srikantan, V.et al., Int J Cancer 84 (1999) 331–335; MacGrogan, D. and Bookstein, R.,Seminars in Cancer Biology 8 (1997) 11–19; Verma, R. S. et al., CancerInvestigation 17 (1999) 441–447) and breast cancer (Bilanges, B.,Oncogene 18 (1999) 3979–3988; Chappell, S. A. et al., British J Cancer75 (1997) 1324–1329; Devilee, P., et al., Oncogene 6 (1991) 1705–1711;Noviello, C. et al., Clin Cancer Research 2 (1996) 1601–1606; Fujii, H.,et al., Genes, Chromosomes & Cancer 16 (1996) 35–39). The location ofTHW gene on chromosome 6q is within the interval D6S472-D6S453.

Loss of heterozygosity (LOH) in the THW gene can be detected accordingto the state of the art preferably with POR using microsatellite markerslocated in the above-mentioned interval. For the detection of LOH andstatus of microsatellite instability (MSI) determination primers(markers) from this interval and/or primers from the flanking intervalsD6S434-D6S302 and D6S453-D6S311 can be used. Microsatellite markerswithin the intervals mentioned can be found in the Genome Database GDB.MSI detection can occur, using primers from the above-mentioned flankingregions, according to known methods as described, for example, by de laChapelle, A., Eur. J. Hum. Genet. 7 (1999) 407–408 and Potocnik U. etal., Pflugers Arch 439 (3.sup.rd Suppl.) 2000, R47-9. Methods of LOHdetection are described, for example, by Friedrich, M. G., et al., J.Urol. 163 (2000) 1039–1042; Sugano, K., et al., Genes, Chromosomes &Cancer 15 (1996) 157–164; Chen, Y. H., et al., J. Pathol. 177 (1995)129–134; Hahn, M., et al., BioTechniques 18 (1995) 1040–1047;Lopez-Crapez, E., et al., BioTechniques 17 (1994) 1072–1 074, 1076;Dockhorn-Dworniczak, B., et al., Virchows Arch. 424 (1994) 337–342;Gruis, N. A., et al., Br. J. Cancer 68 (1993) 208–213; Merlo, G. R., etal., BioTechniques 11(1991)166–168, 170–171.

Preferably, higher molecular weight DNA is isolated from paraffinsections or from other tissue samples. Analysis is performed by PCRusing oligonucleotides flanking polymorphic microsatellite markerscontaining dinucleotide repeats.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention. It isunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the invention.

SEQ ID NO:1: cDNA and amino acid sequence of THW. SEQ ID NO:2: Aminoacid of THW. SEQ ID NO:3: Primer 312rev1. SEQ ID NO:4: Primer 312f6. SEQID NOS:5–10: Primer

DESCRIPTION OF THE FIGURES

FIG. 1 Differential expression of the THW gene in cell lines 530 andNMCL-1.

-   -   RNA was size-separated on a 1% agarose-formaldehyde gel and        hybridized to α-³²P-labelled probe derived from THW cDNA as        described in the Material and Methods section.    -   Lane a: cell line 530; lane b: cell line NMCL-1

FIG. 2 Putative topology of the gene product (SEQ ID NO: 2) of the THWgene showing (from top to bottom) the extracellular, transmembrane andintracellular domains. The topology prediction is based on computerprogram TMHMM.

FIG. 3 Expression of mRNA for the THW gene in human melanoma cell lineswith different metastatic capacity.

-   -   Northern blot was performed as described in the Materials and        Methods section. The blot was hybridized with a α-³²P-labelled        probe derived from the THW gene. In each lane a different        melanoma cell line is shown.    -   Lanes: a, BLM; b, MV3; c, NMCL-1 d, IF6m; e, Mel57; f, M14; g,        MV-1; h, IF6; i, 530.

FIG. 4 Expression of the THW gene in breast carcinoma cell lines.

-   -   The cell lines are derived from normal breast epithelium (HMEC),        primary mammary carcinoma (AR and WA), bone marrow        micrometastases (1590, HG15 and KM22) and from ascites fluid        (KS). Northern blotting was performed as described in the        Materials and Methods section and the blot hybridized with an        α-³²P-labeled probe derived from THW cDNA.    -   Lane a: HMEC; b, AR; c,WA; d, 1590; e, HG15; f, KM22; g, KS.

FIG. 5 Expression of the THW gene in pancreas carcinoma cell lines.

-   -   Cell line K2 is derived from a pancreas primary tumor and three        cell lines (K3, K13, K16) are derived from metastases at        different sites (K3 from the mesenterium, K13 from Porta hepatis        and K16 from lung).    -   Northern blotting was performed as described in the Materials        and Methods section and the blot hybridized with an        α-³²P-labeled probe derived from THW cDNA.    -   Lane a: K2; b, K3; c, K13; d, K16;

FIG. 6 Expression of THW in selected tumors and their correspondingnormal tissues.

-   -   Northern blot analysis was performed as described in the        Materials and Methods section. The blot was hybridized to an        α-³²P-labeled cDNA derived from THW cDNA.    -   Lane a: normal ovary; b: ovarian tumor; c: normal cervix; d,        cervical cancer; e: normal uterus; f: uterine tumor; g: normal        breast; h: breast tumor.

FIG. 7 Expression of the THW gene in selected tissues and cell lines.

-   -   The Multiple Tissue (MTE™) Array (Clontech, Palo Alto, Calif. )        was hybridized with an α-³²P-labeled probe corresponding to THW        cDNA according to the recommendations of the manufacturer. E6        corresponds to 1 μg poly A⁺RNA from cell lines AR, H6        corresponds to 0.1 μg poly A⁺RNA from cell line AR. The coding        is revealed above the blot.    -   A1=whole brain; A2=cerebellum, left; A3=substantia nigra;    -   A4=heart; A5=esophagus; A6=colon, transverse; A7=kidney,    -   A8=lung; A9=liver; A10=leukemia, HL-60; A11=fetal brain;    -   A12=yeast total RNA.    -   B1=cerebral cortex; B2=cerebellum, right; B3=accumbens nucleus;        B4=aorta; B5=stomach; B6=colon, descending;    -   B7=skeletal muscle; B8=placenta; B9=pancreas; B10=HeLaS3;    -   B11=fetal heart; B12=yeast tRNA.    -   C1=frontal lobe; C2=corpus callosum; C3=thalamus; C4=atrium,        left; C5=duodenum; C6=rectum; C7=spleen; C8=bladder;    -   C9=adrenal gland; C10=leukemia, K-562; C11=fetal kidney,    -   C12=E.coli rRNA.    -   D1=parietal lobe; D2=amygdala; D3=pituitary gland; D4=atrium,        right; D5=jejunum; D7=thymus; D8=uterus; D9=thyroid gland;    -   D10=leukemia, MOLT-4; D11=fetal liver; D12=E.coli DNA.    -   E1=occipital lobe; E2=caudate nucleus; E3=spinal cord;    -   E4=ventricle, left; E5=ileum; E7=peripheral blood leukocyte;    -   E8=prostate; E9=salivary gland; E10=Burkitt's lymphoma, Raji;    -   E11=fetal spleen; E12=Poly r(A).    -   F1=temporal lobe; F2=hippocampus; F4=ventricle, right;    -   F5=ilocecum; F7=lymph node; F8=testis; F9=mammary gland;    -   F10=Burkitt's lymphoma, Daudi; F11=fetal thymus; F12=human        C_(o)t-1 DNA.    -   G1=paracental gyrus of cerebral cortex; G2=medulla oblongata;    -   G4=interventricular septum; G5=appendix; G7=bone marrow;    -   G8=ovary; G10=colorectal adenocarcinoma SW480; G11=fetal lung;        G12=human DNA 100 ng.    -   H=pons; H2=putamen; H4=apex of the heart; H5=colon, ascending;        H7=trachea; H10=lung carcinoma, A549;    -   H12=human DNA 500 ng.

EXAMPLE 1 Determination of the Tumorigenic and Metastatic Capacity

Cell line 530 was derived from a surgically removed human melanomametastasis as described previously (van Muijen, G. N. P. et al., ClinExp Metastasis 9 (1991) 259–272; Versteeg, R. et al., EMBO J 7 (1988)1023–1029). Cell line NMCL-1 was also derived from a human cutaneousmelanoma metastasis. Both cell lines were grown as monolayers in cultureflasks in Dulbecco's modified Eagle medium supplemented with 10% fetalcalf serum, glutamine, penicillin G and streptomycin. To determine thetumorigenic and metastatic capacity of the 530 and NMCL-1 cell lines,tumor cells were harvested from subconfluent cultures by 2 min treatmentwith 0.25% trypsin and 0.02% EDTA. After washing with serum-containingmedium, the cells were suspended in PBS and 5×10⁶ cells were inoculatedsubcutaneously (s.c.) into the lateral thoracic wall of BALB/c athymicnude mice, which were bred in the nude mouse facility of the CentralAnimal Laboratories, University of Nijmegen, The Netherlands. The micewere inspected twice a week for local tumor growth and generalcondition. Two groups of five mice each were injected for each cellline. The mice were killed when signs of illness or respiratory distresswere noted. Mice that remained healthy were killed 3–4 months afterinoculation. Microscopic inspection for the detection of lung metastaseswas performed on paraffin sections from at least 3 different levels ofthe lungs. Cell line 530 showed tumor take in 8 out of 10 inoculatedmice. On microscopic inspection of the lungs, no metastases were foundin any of these mice. Cell line NMCL-1 showed s.c. tumor growth in all10 inoculated mice. In contrast to cell line 530, the NMCL-1 cell lineshowed extensive lung metastases in all mice inoculated with this cellline.

EXAMPLE 2 Differential Display PCR

Differential Display polymerase chain reaction (DD-PCR) was performedaccording to the method described by Liang and Pardee (Liang, P., andPardee, A. B., Science 257 (1992) 967–971; Liang, P., et al., Cancer Res52 (1992) 6966–6968) using the RNA image kits (GenHunter Corp.,Brookline, Mass.) according to the manufacturer's recommendations. TotalRNA was isolated from 530 and NMCL-1 cells by using RNeasy® Midi Kit(Qiagen, Del.). Elimination of contaminating traces of DNA from totalRNA sample was performed by digestion at 37° C. for 30 min withRNase-free DNase I using the MessageClean® Kit (GenHunter Corp.,Brookline, Mass.). DNA-free total RNA (0.2 μg) from 530 and NMCL-1 cellswas used as a template for first strand cDNA synthesis in the presenceof 3 different one-base anchored H-T₁₁M primers, 1×reverse transcriptasebuffer [125 mM Tris-Cl, pH 8.3, 188 mM KCl,7.5 mM MgCl₂, 25 mMdithiothreitol (DDT)] and 250 μM dNTP mix. The solution was heated to65° C. for 5 min and cooled to 37° C. for 10 min and then 200 units ofMoloney murine leukemia virus (MMLV) reverse transcriptase was added.After incubation at 37° C. for 1 h, the reaction was terminated byincubation at 75° C. for 5 min. The PCR procedure was performed insolution containing 0.1 volume of reverse transcription reactionmixture, 10 μM of the respective one-base anchored H-T₁₁M primer, 2 μMarbitrary 13-mer primer, 1×PCR buffer [100 mM Tris-Cl, pH 8.4, 500 mMKCl, 15 mM MgCl₂, 0.01% gelatin], 25 μM dNTP, 10 μCi [α-³⁵S]dATP, and 10units of AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, Conn.). The PCRincluded a total of 40 cycles at 94° C. for 30 s, 40° C. for 2 min, 72°C. for 30 s, and finally 5 min at 72°C. After adding 2 μl loading bufferto 3.5 μl of each sample, the PCR products were heated at 80° C. for 2min and then loaded on a denaturing 5% polyacrylamide sequencing gel forelectrophoresis. The dried gel was exposed to Kodak BioMax® MR film for48 h at room temperature and the autoradiogram was analyzed with respectto differentially expressed genes. The reaction displaying uniquefragments in one of the two cell lines was subsequently confirmed byrepeating reverse transcription and PCR. Unique bands reproduciblydisplayed in two independent DD-PCR reactions were excised from thedried gel and the cDNA was eluted from the gel by soaking the gel slicein 100 μl of H₂O for 10 min and then boiling for 15 min. The cDNA wasrecovered by ethanol precipitation in the presence of 3M NaOAc and 50 μgglycogen as carrier and redissolved in 10 μl of H₂O. Four μl of elutedcDNA was reamplified in a second PCR using the same 5′- and 3′-primersand conditions described above except for dNTP concentrations of 20 μMand no radioisotope was included. The amplified PCR fragments obtainedwere analyzed on a 1.5% agarose gel, then purified using the QIAquick®Gel Extraction kit (Qiagen, Hilden, Del.) and used as probes forNorthern analysis.

EXAMPLE 3 Sequencing and Characterization of THW Gene

Cloning of DD-PCR Fragments

Northern analysis was first performed using hybridization probesgenerated directly from PCR reamplification. Those amplified PCRfragments detecting differentially expressed mRNAs on a Northern blotwere subcloned into the PCR 2.1-TOPO vector by the Topo TA Cloningsystem (Invitrogen, San Diego, Calif.). Subdoned fragments were isolatedusing the Qiagen plasmid kit (Qiagen) and again used as probes forNorthern analysis to verify differential expression.

DNA Sequencing of Subcloned DDRT-PCR Fragments

Those subdoned fragments corresponding to mRNAs with differentialexpression were sequenced directly after subcloning into the Topo TAcloning vector (see above) using the Dye Terminator Cycle Sequencing kit(Applied Biosystems, Foster City, Calif.). The nucleotide sequence datawere analyzed for homologies with known genes of EST's in the currentDNA databases.

RT-PCR

An RT-PCR was performed to identify the 5′-extended region of the cDNAshowing differential mRNA expression. The resulting 301 bp clone fromthe Differential Display analysis was run against EMBL Database. Fulllength cDNA could be obtained from the consensus sequence of 11homologous, overlapping ESTs (Accesion Numbers: W93394, AA480373,AA610151, AA468385, R 82584, AA159815, AA159565, AI814625, AI740811,AI8300092, AI694126). The primers were designed from the resultingconsensus sequence. RT-PCR was performed with the C. therm PolymeraseOne-Step RT-PCR System according to the manufacturer's instructions(Roche). The system is composed of an enzyme mix containing the Klenowfragment of DNA polymerase from Carboxydothermus hydrogenoformans andthe thermostable Taq polymerase.

First Strand Synthesis

190 ng poly A⁺ RNA of the cell-line 530 was reverse transcribed with theprimer 312revl (5′AAATCCCCGAATTCTCCTGTGG3′, SEQ ID NO:3) with a finalconcentration of 0.3 μM. DMSO (7%) was added in order to eliminatesecondary structures at the 5′-end of the RNA due to a very high GCcontent (>75%). Incubation followed for 30 min at 65° C.

PCR

The PCR was performed in the same tube as the RT reaction. The firststrand cDNA was used as template for the following PCR using the primer312revl and primer 312f6 (5′ACCCGCTCCGCTCCGCTC3′, SEQ ID NO:4) withfinal concentration of 0.3 μM each. PCR conditions were as follows:35×94° C.—30 sec, 69° C.—30 sec, 72° C.—60 sec. The resultingPCR-fragment was subcloned and analyzed.

Northern Blot Analysis

Poly A⁺ RNA was isolated from total RNA using the Oligotex® mRNA MiniKit (Qiagen, Hilden). Parallel lanes of poly A⁺ RNA from human melanomacell lines, human breast carcinoma cell lines and pancreas carcinomacell lines (1 μg of each cell line) were size-separated on a denaturing1% agarose formaldehyde gel. Blotting to BrightStar-Plus™ (Ambion Inc.,Austin, Tex.) positively charged nylon membrane was done by capillarydownward transfer. After UV-crosslinking (Stratagene UV Stratalinker™2400) blots were hybridized to [α-³²P]dCTP—labeled DD-PCR productsprepared by random decamer (10-mer) priming and labeled to a specificactivity of 2×10⁹ cpm/μg using the Strip-EZ™ DNA Kit (Ambion Inc.,Austin, Tex.). Prehybridization (0.5 h) and hybridization withradioactive probes overnight were performed in ExpressHyb™ HybridizationSolution (Clontech) at 68° C. Membranes were washed in Solution 1(2×SSC, 0.05% SDS) at room temperature for 30–40 min with continuousagitation and several replacements of the wash solution 1 followed by awashing step with solution 2 (0.1×SSC, 0.1% SDS) at 50° C. for 40 minwith one change of fresh solution. The membranes were then exposed toCronex™ Medical X-Ray Films (Sterling Diagnostic Imaging Inc., USA) at−80° C. for 3 to 72 h. Equal loading and transfer of mRNA to themembrane was assessed by hybridizing the blots with ³²P-labeled β-actincDNA.

Multiple Tissue Expression Array and Human Tumor Panel Blot

To examine the tissue-specific expression of the THW gene, thedistribution of THW mRNA in different human tissues and cell lines wasanalyzed by Northern blot analysis using a Multiple Tissue Expression(MTE™) Array (Clontech, Palo Alto, Calif.) and a Human Tumor Panel Blot(Invitrogen). The Tumor Panel Blot contains a panel of tumor RNA fromdifferent tissues, normal RNA is run adjacent to the tumor RNA. The MTEblot contains 76 tissue-specific polyA⁺ RNAs. The different blots wereprobed with ³²P-labeled THW cDNA probe. Equal loading of mRNA wasverified by rehybridizing the different blots with ³²P-labeled β-actincDNA.

Sequence Analysis

The PSORT II computer program (Human Genome Center, Institute forMedical Science, University of Tokyo, Japan) was used for the predictionof protein sorting signals and localization signals in the amino acidsequence. The TMHMM (v.0.1) computer program of the Center forBiological Sequence Analysis, Department of Biotechnology (The TechnicalUniversity of Denmark) was used for the prediction of transmembranehelices and their orientation in the membrane.

The cDNA corresponds to 1890 nts with a potential ORF of 193 aa. Apolyadenylation signal was identified at nts 1855–1861.Bioinformatic-based analysis suggested a four-transmembrane receptortopology (FIG. 2). Topology of THW corresponds to a receptor with twoextracellular domains (45 aa and 18 aa), four transmembrane domains(19aa, 24aa, 23aa and 23aa) and three cytoplasmic domains (12aa, 6aa and24aa). Homologies were found on sequence comparisons with nucleotidesand proteins described in WO 98/39448; WO 99/54461; WO 98/55508; and WO99/61471.

The correlation between the metastatic capacity of human melanoma cellsin the nude mouse system and the mRNA steady-state level of the THW geneis summarized in FIG. 3. Highest steady-state level was found in thenon-metastatic cell line 530 (FIG. 3, lane i), the lowest steady-statelevel was found in the highly metastatic cell lines BLM and MV3, asshown in lanes a and b, and intermediate levels corresponded to celllines IF6m, NMCL-1, Mel57, M14, MV-1 and IF6 (van Muijen, G. N. P. etal., Clin Exp Metastasis 9 (1991) 259–272; Versteeg, R. et al., EMBO J 7(1988) 1023–1029; van Muijen, G. N. et al., Int J Cancer 48 (1991)85–91; van Groningen, J. et al., Cancer Res 55 (1995) 6237–6243;Weterman, M. A. J. et al., Cancer Res 52 (1992) 1291–1296), all of whichare cell lines with intermediate potential for metastasis. These resultsclearly establish a correlation between down-regulation of expression ofthe THW gene and the metastatic potential of human melanoma cells in thenude mouse system.

These investigations were extended to selected mammary carcinoma celllines and their non-malignant equivalents as shown in FIG. 4. Cell linesincluded the normal human mammary gland epithelial cells (HMEC), celllines derived from primary tumors (WA and AR), three cell lines derivedfrom bone marrow micrometastases of mammary carcinoma (KM22, HG15 and1590) and one cell line derived from ascites (KS). With the exception ofcell line WA (FIG. 4, lane c), all other mammary carcinoma cell lines(FIG. 4, lanes a, b, c, d, f) exhibited significant (10-fold)down-regulation of the steady-state level of the mRNA of the THW gene.These results indicate that down-regulation of expression of the THWgene plays a role in the pathogenesis of mammary carcinoma as well.Similar results were found within a panel of cell lines established froman orthotopic xenograft mouse model. One cell line (K2) was derived froma primary tumor and three cell lines were derived from metastases atdifferent sites (K3 from the mesentery, K13 from the porta hepatis, K16from the lungs). As shown in FIG. 5, THW mRNA was downregulated in allthree cell lines derived from metastases (K3, K13, K16) compared withthe cell line derived from the primary tumor (K2).

The steady-state level of THW mRNA in several tumor samples (breast,uterus, cervix and ovarian carcinomas) was compared with thecorresponding normal tissues as outlined in FIG. 6. In breast, uterineand cervical carcinomas dramatically reduced mRNA levels of THW werefound in comparison with the corresponding normal tissues; in ovariancarcinoma, no signal was detected in either normal or tumor tissue.These experiments indicate downregulation of the THW gene in some solidtumors.

Expression of the THW gene in normal and selected fetal tissues as wellas selected tumor cell lines was investigated by making use of multipletissue expression array (MTE™, Clontech) as shown in FIG. 7. THW wasexpressed in tumor cell lines such as HeLa, colorectal adenocarcinomacell line SW480 and lung carcinoma cell line A549, but was very weaklyexpressed in erythroleukemic cell line K562 and not expressed in otherhematopoietic tumor cell lines such as leukemia cell lines Molt4,leukemia HL-60 and Burkitt's lymphoma cell lines Raji and Daudi. Noexpression could be detected in peripheral blood leukocytes and onlyvery weak expression was found in brain and its compartments, skeletalmuscle, spleen, lymph node, bone marrow, testis and ovary. Intermediateexpression was found in the heart and its compartments, gastrointestinalorgans, kidney, thymus, lung, bladder, placenta, uterus, liver,pancreas, adrenal gland, thyroid gland, salivary glands, prostate andmammary glands; high expression was found in the esophagus and trachea.THW was expressed in fetal heart, kidney, liver, thymus and lung andonly very weakly in fetal brain and spleen. THW is thus almostubiquitously expressed with exception of peripheral blood leukocytes.The receptor is ubiquitously expressed with the exception of peripheralblood leukocytes (FIG. 7).

The tumor-suppressor function of THW is therefore characterized by thefollowing findings:

-   a) Down-regulation of the THW gene in metastasizing human melanoma    cells compared with intermediate and non-metastasizing cell lines in    the nude mouse system (FIG. 3)-   b) Down-regulation of the THW gene in mammary carcinoma cell lines    compared with human mammary gland epithelial cells (FIG. 4)-   c) Down-regulation of the THW gene in pancreas cell lines derived    from metastases compared with a cell line derived from a primary    tumor (FIG. 5).-   d) Down-regulation of the THW gene in tumor tissue compared with    normal tissue (FIG. 6)

EXAMPLE 4 Detection of LOH

Microsatellites are short sequences (50–300 bp) composed of tandemlyrepeated monomers (1–6 bp) These microsatellites are widespreadthroughout the genome and many of them are highly polymorphic.Polymerase chain reaction (PCR) analysis was used to study the incidenceof allelic loss at 6q24 of various cell lines and tumor biopsies. Forthis propose, oligonuclotides flanking three polymorphic microsatellitemarkers were used. All of them were dinucleotide repeats.

Pairs of normal DNA and autologous tumor DNA (primary melanomas andmetastatic melanoma) from 35 patients were analyzed, using a panel ofthree polymorphic DNA markers localized to the long arm of chromosome 6.There were also analyzed various human melanoma, breast, cervix,prostate and ovary carcinoma cell lines.

LOH is defined as >50% loss in relative peak height of a tumor allelecompared to the normal allele.

We used the following formula to calculate the LOH:

${LOH} = \frac{( {{peak}\mspace{14mu}{height}\mspace{14mu}{of}\mspace{14mu}{normal}\mspace{14mu}{allele}\mspace{14mu}{2/{peak}}\mspace{14mu}{height}\mspace{14mu}{of}\mspace{14mu}{normal}\mspace{14mu}{allele1}} )}{( {{peak}\mspace{14mu}{height}\mspace{14mu}{of}\mspace{14mu}{tumor}\mspace{14mu}{allele}\mspace{14mu}{2/{peak}}\mspace{14mu}{height}\mspace{14mu}{of}\mspace{14mu}{tumor}\mspace{14mu}{allele1}} )}$

Allelic loss is indicated by an LOH value less than 0.5 or higher than2.0.

a) DNA extraction

High molecular weight DNA was isolated from 25 primary melanomas and 10melanoma metastases. The DNA was isolated from (micro)dissected paraffinsections. DNA was also isolated from normal tissue from the samepatients. DNA from tumor cell lines was extracted from pellected cells.The DNA was isolated using the QIAamp® (Qiagen, Del.) DNA Mini Kit.

b) PCR analysis

Fluorescence labeled primers flanking highly polymorphic dinucleotiderepeats at D6S292, D6S1684 and D6S311.

Hetero- Sequence forward Sequence Intervall Locus Marker zygosityModification reverse Product D6S472– D6S292 AFM203za9 0.834tccttcccacctcccttct Taagaactaaagttgcctgttc 106 D6S453 (SEQ ID NO:5) (SEQID NO:6) 5′6-FAM D6S472– D6S1684 AFM360th9 0.8 caactggattcaaaatagatgtcAtggcagcaggctatgt 247 D6S453 (SEQ ID NO:7) (SEQ ID NO:8) 5′HEX D6S453–D6S311 AFM276xf1 0.92 Atgtcctcattggtgttgtg Gattcagagcccaggaagat 259D6S311 (SEQ ID NO:9) (SEQ ID NO:10) 5′HEXPCR conditions were as follows:Set up Master Mix 1:

Component Vol. Final conc. Forward primer 1 μl 300 nM Reverse primer 1μl 300 nM Template DNA X μl 50–100 ng Sterile water, PCR grade Up to 25μlMaster Mix 2:25 μl High Fidelity PCR Master (Roche Diagnostics GmbH, DE)

Pipet both mixes together. PCR was carried out using the followingcycles: 2 min 94° C. and 40×94° C.—30 sec, 55° C.—30 sec, 72° C.—30 secwith a final extension of 7 min at 72° C. The three primer pairsgenerate flourescence labeled PCR products for analysis on the ABI PRISM310 Genetic Analyzer with the GeneScan Analysis Software (PE AppliedBiosystems).

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1. A process to screen a test sample containing cells for potentials todevelop tumor, tumor progression or metastasis, against a base sample,the process comprises the following steps: (a) incubating the testsample and the base sample under hybridization conditions with a nucleicacid probe selected from the group consisting of: (i) a nucleic acidsequence of SEQ ID NO:1; (ii) a nucleic acid sequence which iscomplementary to any nucleic acid sequence of (i); (iii) a nucleic acidsequence which hybridizes under stringent conditions with the sequenceof (i); and (iv) a nucleic acid sequence which hybridizes understringent conditions with the sequence of (ii); (b) determining theamount of hybridization of the test sample and the base sample with saidprobe; and (c) comparing the amount of hybridization of the test sampleto an amount of hybridization of said base sample: wherein if the amountof hybridization of the test sample is less than the amount ofhybridization of the base sample, the cells in the test sample have agreater potential for developing or progressing tumors or metastasisthan the cells in the base sample.
 2. The process according to claim 1wherein the test sample originates from or contains human cells.
 3. Theprocess according to claim 1 wherein the base sample originates fromnon-tumor cells obtained from human cells.
 4. The process according toclaim 1 wherein the hybridization proceeds under stringent conditions.